Aire regulates central T cell tolerance by controlling thymic medullary epithelial cell (MEC) expression of a battery of transcripts encoding peripheral-tissue antigens. From early on, Aire was recognized to be a transcriptional regulator, but it soon became apparent that it does not operate like a conventional transcription factor, i.e. binding to promoters and inducing initiation of transcription. Studies completed during the last funding-cycle demonstrated that Aire-induced genes occur as intra- or inter-chromosomal clusters within individual cells, that Aire participates in multiple multi-protein complexes, and that it preferentially localizes to and activates so-called ?super-enhancers.? Super-enhancers (a.k.a ?stretch? or ?serial? enhancers) are extended regions of chromatin that are over-loaded with general and cell-type-specific transcription factors, and are thought to serve as depots for coordinate and efficient delivery of these factors to targeted promoters via chromatin looping. In addition, preliminary data documented herein indicate that Aire associates, directly or indirectly, with cohesin, one of the major orchestrators of long-range chromatin interactions (in collaboration with CTCF and/or mediator). Thus, the overall goal of this proposed project is to determine how Aire integrates into the three-dimensional organization of chromatin. This goal will be addressed via three Specific Aims: ? To determine whether Aire associates with proteins known to orchestrate 3D chromatin interactions: CTCF, cohesin, NIPBL and mediator. Experiments under this Aim will employ primarily biochemical approaches to determine whether Aire associates with the three major 3D-chromatin-organizing elements (and a cohesin-loader, NIPBL). ? To integrate Aire?s distribution along MEC chromatin with those of CTCF, cohesin and mediator; to determine how Aire impacts CTCF/cohesin/mediator placement and, vice versa, how cohesin influences Aire?s placement. This series of experiments will exploit recent improvements in ChIP-seq (and other whole-genome) technologies to map the binding sites of the three chromatin organizers in relation to Aire binding and other landmarks ? in wild-type, Aire-knockout and inducible Smc1-knockdown mice ? To define the relationship between Aire and three-dimensional chromatin organization. This set of experiments will use HiC technology to generate whole-genome interaction maps for MEC chromatin from Aire+/+ and Aire-/- mice. Successful completion of these studies will bring our understanding of Aire control of T cell tolerance to a new level of molecular understanding, and is likely to resolve several of the outstanding conundrums related to Aire function. The Aire story continues to intrigue!